CASE CLOSED … what really happened in the 2001 anthrax attacks?

Archive for July 8th, 2009

* Dr. Paul Keim answers questions about the anthrax investigation, some from CASE CLOSED blog participants

Posted by Lew Weinstein on July 8, 2009

why the FBI failed to solve the 2001 anthrax caseCASE CLOSED

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Dr. Paul Keim answers questions

about the anthrax investigation,

some from CASE CLOSED blog participants

KeimPaul Keim, Ph.D., is a Regents Professor of Biology at Northern Arizona University and the Division Director of Pathogen Genomics at the Translational Genomics Research Institute (TGen). Keim serves on the Scientific Working Group for Microbial Forensics (FBI) and the National Science Advisory Board for Biodefense to advise the U.S. government on issues of science and policy.

On July 6, 2009, Paul Keim answered selected viewer questions about the anthrax letter attacks and microbial forensics in general. Excerpts from these Q & A follow; the entire Q & A is found in the comment below.

Q: What are the limitations of using microbial forensics for attribution purposes? Are policymakers fully aware of these limitations? Jonathan B. Tucker, Washington, D.C.

A: … “The laboratory Ames strain can be precisely identified and differentiated from all other types of Bacillus anthracis, including those very close relatives isolated from the same geographic region of Texas.” This is a long ways from concluding that letter spores came from USAMRIID, but it is a start and eliminates a lot of possibilities. “Exclusion” due to a lack of a match is one of the most powerful conclusions that can be drawn in DNA fingerprinting analysis. In the Amerithrax case, the scientific evidence (morph typing) tying the letter spores to the RMR-1029 appears strong. The FBI repository of Ames cultures was extensive, and only cultures derived from RMR1029 or that culture itself have all four morphs. While this scientific conclusion excludes a very large number of possible perpetrators, it still doesn’t directly attribute the crime to an individual. I understand that there was more than one person with access to RMR-1029 spore preparation. I don’t know how many individuals had access, and I can only speculate that it could have been quite a few (10? 50? 100?). This limitation is well understood by the scientists involved but may or may not be understood by the public or policymakers.

Q: Did the FBI or U.S. Department of Justice consult with any genetics expert when they were asked in October 2001 whether it was okay for the [B. anthracis strains held at] Iowa State University and USDA Ames (at the strip mall) to be destroyed? Ross Getman, Syracuse, New York

A: I was not consulted nor am I familiar with the actual steps that lead to the destruction of these materials. During that time period (late 2001), I was doubtlessly the most engaged microbial geneticist working with the FBI. If I wasn’t consulted, then probably none others were either. Additional Commentary:  It is hard to understand why this destruction was done and why it was allowed to occur. Clearly someone in Iowa panicked at being in the media bright lights and wanted to get rid of the material. If this was really authorized by the FBI, who that authority was has not been released, to my knowledge. If the investigation had eventually lead back to Iowa, this would have been viewed as destruction of evidence and obstruction of justice. Now that would have been a public relations nightmare!

The following are several of the many questions proposed at my CASE CLOSED blog, where a vibrant discussion of issues related to the anthrax investigation is active every day. Lew Weinstein, Collioure, France:

Q1: Did the 1,070 samples examined by the morphotype analysis accurately reflect the actual number of anthrax samples held in the U.S. in the 2001 timeframe?

A: This would not be representative of all the Bacillus anthracis samples, but rather Ames strain stocks. I don’t know how many B. anthracis cultures existed in the U.S. in 2001, but many more than this. The Ames strain is only one of many different identifiable strains.

Q2: Did the process of self-submission of anthrax samples by labs and individuals (per the FBI methodology) result in a reliable representation of the Ames samples that may or may not have existed prior to 2001?

A:  Presumably, the 1,070 collection represents all the Ames cultures in the U.S. plus some from around the world. First, the Department of Justice subpoenaed all the U.S. anthrax labs for our inventory records with special attention to the Ames strain cultures. Next, the DOJ subpoenaed all U.S. laboratories that had the Ames strain to provide a sampling (a portion) of their Ames cultures. For example, my lab had only seven different Ames stocks, which we sampled and sent in duplicate to the FBI.

Q3: Did the FBI use any type of proficiency testing for their genetic analysis?

A: Definitely yes. Extensive proficiency testing was performed both prior to and during the genetic analysis at Northern Arizona University. The initial determination from the first Florida sample was done using normal research controls and standards, but soon afterwards very rigorous forensic-level protocols were implemented. There was no time for proficiency testing for the first analysis on the Robert Stevens isolate, as we had only a few hours notification. Immediately afterwards, the FBI forensic scientists began to review our procedures and help us to institute forensic-level operating protocols, including proficiency testing. Once these were in place, original analyses were repeated with the same result.

Note that there were no forensic standards for microbes and that we had to adapt the ones developed for human DNA testing to our new anthrax tests. This is a topic of great debate as to what the standards should be for a new technology.

Q4: Was the Bacillus subtilis contaminant found in the first batch of letters genetically identical to any forensic evidence collected from any lab? Also, was that contaminant tested against strains from Dugway Proving Grounds?

A: Disclaimer: My lab did not work on the B. subtilis contaminant, so what I am relating is secondhand knowledge. However, it is based upon my conversations with government experts and my participation in the press conferences and the public ASM symposium. Details of the contaminant were discussed in these forums. Background: The question refers to the non-hemolytic (B. anthracis is hemolytic) bacterial contaminant found in some of the letters. It proved to be a spore forming Bacillus subtilis bacterium. As the name implies, this is somewhat related to Bacillus anthracis and shares many biological properties. But it is not a disease causing organism, and there are many, many different types found in the environment. It would not be surprise to me to find a novel B. subtilis on my computer keyboard. The government investigators thought that perhaps this contaminant could be used to trace back the spore preps to a particular laboratory. If the contaminant was a common laboratory strain of B. subtilis, this might have been possible. In the end, this apparently proved impossible because this contaminant was different from any known lab strains. Likewise, Dugway Proving Grounds labs were intensely investigated, and I would assume that any Bacillus strains at that facility would have been investigated. Again, no lab strains matched the contaminant using DNA analysis.

Three questions from Marcia Ann Chambers, Topeka, Indiana

Q1: The mailed spores were compared to ancestral Ames (genotype62) and determined to be identical to it. In what lab and under what circumstances was the ancestral Ames stored?

A: The ancestral Ames culture was stored at USAMRIID until it was discovered during the Amerithrax investigation. It is the oldest known archival Ames culture discovered in the investigation and dates from early 1981 (see: Ravel et al. 2009 J. Bacteriology 191:445-6.).

Q2: When testing other sources to compare to the mailed spores, did you look for colony morphology differences?

A: The morphs themselves were useful for identifying minor genetic differences in the letter spores, but it would have been very tedious and unreliable to depend upon visual analysis of colony morphology across the entire repository or across all the evidence. In addition, the colony morphological differences can be subtle and subjective. (A lot of credit should be extended to the skilled microbiologists who originally spotted the morphs.) So, the final repository analysis was of the genetic differences controlling the morphological differences. These were genetic tests (PCR) developed after the full genomes of each morph were determined by TIGR [The Institute for Genomic Research]. These tests are rapid,
sensitive, and highly reproducible. The entire process was done in a blinded fashion where the labs used anonymous samples.

Q3: If Iowa State University destroyed its entire collection of anthrax, how can it be eliminated as a source for flask RMR-1029?

A: It is regrettable that ISU destroyed their archival B. anthracis isolates (see my commentary above), as this could have exonerated them. Our DNA analyses would have quickly determined whether they even had the Ames strain in their collection. The reason they were under such scrutiny was due to the misnaming of the Ames strain (see my discussion on this topic above).

Commentary: This is an opinion, but given the mailing location in New Jersey, it is unlikely that an ISU employee could have driven that far to mail the letters without being detected. It is also unlikely that ISU even had the Ames strain, let alone RMR1029. They did not have a vaccine development program, and there were no notable anthrax research teams there at that time. After all the unwarranted media attention, I’m guessing that the government investigated activities at ISU with great diligence and ruled out potential suspects.

NOTE: The entire post from Dr. Keim is contained in the comment below, submitted by DXer and reproduced below.


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